Kinase inhibitors and the use thereof

ABSTRACT

The present invention relates to protein kinase inhibitors and to the use thereof for the treatment of diseases induced by pathological signal transduction cascades.

[0001] The present invention relates to protein kinase inhibitors and tothe use thereof for the treatment of diseases induced by pathologicalsignal transduction cascades.

[0002] Protein kinases are enzymes belonging to the transferases whichcatalyze the transfer of phosphate residues from adenosine5′-triphosphate (ATP) or guanosine 5′-triphosphate (GTP) to proteins. Adistinction is made, according to the amino acid residues to which thephosphate group is transferred, between, for example, proteinserine/threonine kinases, protein hisitidine kinases, protein aspartatekinases or protein tyrosine kinases.

[0003] Protein kinases play a crucial part in regulating the activity ofacceptor proteins (signal transduction cascade). Signals from outsidethe cell are picked up by cell surface receptors such as, for example,receptor tyrosine kinases (RTKs) (Ullrich et al. 1990, Cell, 61:203-212; Fantl et al., 1993, Annu. Rev. Biochem., 62: 453-481).Autophosphorylation of RTKs takes place through the binding of“signal-emitting” molecules or so-called effectors or ligands (Weiss etal., 1997, Curr. Opin. Genet. Div., 7: 80-86). This autophosphorylationpermits the RTKs to interact with other proteins, including so-calledadapter proteins (Robertson et al., 2000, Trends Genet., 16: 268-271).These protein complexes are in turn able to activate other intracellularproteins, leading to a whole chain of protein interactions whereby theoriginally extracellular signal is transmitted from the cell surfaceinto the cell nucleus (Treisman et al., 1996, Curr. Opin. Cell. Biol.,8: 205-215; Tan et al., 1999, Trends Genet., 15: 1456-149). Thetransmitted signal is thus able to influence gene expression, the cellcycle or other important cell functions.

[0004] The effectors of receptor tyrosine kinases include, for example,insulin and many growth factors such as, for example, the growth factorsof the blood platelets (PDGF) or epidermal growth factors (EGF).Receptor tyrosine kinases play an important part inter alia inregulating the formation of new blood vessels (angiogenesis) or newlymphatic vessels (lymphangiogenesis). This entails endothelial cellsfrom pre-existing vessels being stimulated to grow, to proliferate andto extend themselves in order to form new capillary vessels. Particularmention should be made in this connection of the cell surface receptorsVEGFR (vascular endothelial growth factor receptor) and FGFR (fibroblastgrowth factor receptor) and, as effectors, corresponding growth factorsof the VEGF family or FGF family (Korpelainen et al., 1998, Curr. Opin.Biol., 10: 159-164; Malonne et al., 1999, Clin. Exp. Metastasis., 17:1-14). Further well-known examples of natural angiogenic effectors(ligands) are, inter alia, tumor necrosis factor (TNF-α), interleukin 8or the so-called Tie₂ ligand (Malonne et al., 1999, Clin. Expl.Metastasis., 17: 1-14).

[0005] Uncontrolled stimulation of protein kinases may lead topathological processes such as, for example, cancer (Porter et al.,1998, Oncogene, 17: 1343-1352). For example, a genetically modifiedreceptor, i.e. a mutated receptor tyrosine kinase, which constitutivelytransmits, even in the absence of a suitable effector, signals to otherproteins, may lead to the development of cancer. Such activationmutations of RTKs are linked to a large number of human diseases(Robertson et al., 2000, Trends Genet., 16: 268-271). Thus, for example,constitutively active FGF receptors are responsible for a large numberof genetic diseases (Table 1). Incorrect regulation of angiogenesisplays an important part in the progress of a large number of diseaseswhich are listed in Table 2 (Malonne et al., 1999, Clin. Exp.Metastasis., 17: 1-14). Thus, various studies have revealed in the caseof cancers that tumors depend in a critical manner on an adequate bloodsupply. If angiogenesis can be inhibited, tumor growth can also bestopped or even reversed (Zetter et al., 1998, Annu. Rev. Med., 49:407-424). Induction of lymphangiogenesis also plays an important part incancers and filariasis (Skobe et al., 2000, Nature Med., 7: 192-198; Raoet al., 1996, J. Parasitol., 82: 550-556).

[0006] The protein kinase activity of receptor tyrosine kinases can inprinciple be regulated in various ways. Thus, for example, it ispossible to employ antibodies which block the receptor kinase/ligandbinding interaction (Brekken et al., 2000, Cancer Res., 60: 5117-5124;Klement et al., 2000, J. Clinic. Invest., Vol. 105, No. 8: 15-24).Alternatively, the use of soluble extracellular receptor sections forbinding the appropriate ligand in an inactive complex (sequestration) isdescribed by Aiello et al. (1995, Proc. Natl. Acad. Sci, Vol. 92:10457-10461). Both the aforementioned antibodies and the solublereceptor portions have considerable disadvantages, however. Both arerapidly removed from the circulatory system. In addition, the moleculesare very large in both cases, and their ability to penetrate tissue isis very limited. Their preparation, especially that of the antibodies,for pharmaceutical application is very complicated and costly.Furthermore, they represent compounds which may induce an immuneresponse, so that their biological efficacy is greatly reduced orentirely abolished.

[0007] A further possibility for regulating the activity of proteinkinases is through inhibition by substrate-like compounds which competefor example with the natural substrate ATG or GTP for the substratebinding domain. Indolinones able to inhibit receptor tyrosine kinases(RTKS) have been described in this connection. Crystallographic studieson the specific RTK fibroblast growth factor receptor (FGFR) have shownthat the oxindole portion of the indolinones interact with the samebinding site as the adenine ring of the natural substrate ATP(Mohammadi, M. et al., Science, 276: 955-960). However, it chemicalstructure of the substituent on the C3 atom of the oxindole determineswhich RTK activity is inhibited. Some indolinones block the activity ofa single RTK; other inolinones inhibit a wide range of RTKs.

[0008] In order to treat diseases in which the activity of proteinkinases has become pathologically out of control, it is therefore ofenormous medical relevance to provide compounds with which thisuncontrolled activity of surface receptor protein kinases,advantageously receptor tyrosine kinases (RTKs), can be regulated,preferably blocked, in order thus to reduce or even suppress theprogress of the disorders. Diseases in which the activity of proteinkinases has become pathologically out of control may be, for example,types of cancer induced by uncontrolled cell proliferation and/ordisordered apoptosis. Further possibilities are diseases such as, forexample, filariasis or diseases induced by disordered angiogenesisand/or lymphangiogenesis processes (concerning this, see also Table 2).It is also desirable in this connection to provide compounds which, viathe natural activity or pathologically altered (constitutive) activityof protein kinases, directly block angiogenesis and/orlymphangiogenesis, so that on the one hand the blood supply to the tumoris lessened or even stopped, leading to cessation of tumor growth ordeath of the pathological tissue, and on the other hand metastastis oftumor cells is prevented.

[0009] This object is achieved in an advantageous manner by the presentinvention.

[0010] The present invention relates to compounds which inhibit theactivity of protein kinases which are involved in uncontrolled cellproliferation and/or disordered apoptosis of cells and/or angiogenesisand/or lymphangiogenesis and/or angiogenesis- and/orlymphangiogenesis-dependent disorders and/or filariasis and represent aderivative, substituted on the C3 atom, of indolin-2-one, selected fromthe group of compounds of the general structural formula

[0011] or salts of the compounds I)-III).

[0012] These compounds are also referred to in the course of thedescription as MAE87 (=structural formula I), MAE106 (=structuralformula II), MAZ51 (=structural formula III). The present inventionfurther relates to a compound called MAZ51-2 which, as a salt of thecompound MAZ51, represents a more soluble variant of MAZ51 withotherwise comparable properties.

[0013] In an advantageous variant of the present invention, thecompounds block the activity of receptor tyrosine kinases which areinvolved in angionesis and/or lymphangiogenesis. In a further variant,the compounds of the invention, singly or in combination thereof,inhibit the activity of receptor tyrosine kinases from the group ofVEGFR-3 (vascular endothelial cell growth factor receptor), VEGFR-2,Tie2, EGFR (epidermal growth factor receptor), ErbB2, IGFR and FGFR1(fibroblast growth factor receptor). An advantageous variant of theinvention includes compounds which block the activity of the cellsurface receptor VEGFR-3.

[0014] For the purposes of the invention, the compounds inhibit not onlythe natural activity of protein kinases but also the activity ofpathologically altered protein kinases, i.e. of protein kinases whoseactivity has become pathologically out of control. The protein kinasesmay be, for example, permanently (constitutively) stimulated. This mayhave been induced by altered properties in the interaction behavior withligands (effectors), or other protein components or an alteredautostimulation of the RTKs.

[0015] Pathologically altered protein kinase variants mean for thepurposes of the present invention for example protein kinases which,owing to alterations at the nucleic acid and/or protein level, bringabout uncontrolled cell proliferation and/or disordered apoptosis ofcells and/or lead to disordered angio-genesis and/or lymphangiogenesisand to the diseases connected therewith (see Table 2) and/or areinvolved in the disease filariasis. Alterations at the nucleic acidlevel mean mutations according to the invention, for which for exampledeletions, insertions or exchanges of one or more nucleotides are to beunderstood. Alterations at the protein level mean deletions, insertionsor exchanges of one or more amino acids.

[0016] The principle of action of the compounds of the invention isbased on their simulation of the natural substrate of the proteinkinases, for example ATP. This means that they act as ATP analogs as itwere. This involves them binding with their oxindole portion to the samedomain of the protein kinases to which the adenine ring of ATP naturallybinds. The compounds of the invention and the natural substrate thuscompete for the substrate binding site, i.e. the compounds of theinvention displace the ATP from the substrate binding site. For thisreason, the protein kinases are unable to catalyze any transfer of aphosphate group to a target molecule, and are thus no longer able toexercise their intended function as kinase. The compounds of theinvention are thus by reason of their structure able not only generallyto inhibit the activity of protein kinases but also to treat humandisorders induced by mutated (constitutively) active RTKs. It isconceivable in this connection to use each of the compounds singly, butalso combinations thereof.

[0017] The compounds of the invention have the advantages that they aresmall molecules exhibiting very good cell and tissue penetrance. Theyare also very suitable for administration to the patient, preferablyorally. In addition, preparation of the compounds for pharmaceutical useis simple and cost-effective even on the industrial scale. It ismoreover particularly advantageous that these compounds do not induce animmune response.

[0018] The present invention also relates to compositions comprising atleast one compound of the invention of the aforementioned type for thetreatment of disorders in the development, progress, alleviation and/orcure of which naturally occurring and/or pathologically altered proteinkinases are involved. This includes according to the inventioncompositions for inhibiting uncontrolled proliferation and/or inducingapoptosis of cells and/or for inhibiting angiogenesis and/orlymphangiogenesis comprising at least one compound of the aforementionedtype. The compositions of the invention comprising at least one compoundof the invention are also suitable for the treatment of angio-genesis-and/or lymphangiogenesis-dependent disorders (as listed for example inTable 2) and/or filriasis. The aforementioned compositions are furtherdistinguished by comprising at least one compound of the type accordingto the invention in a concentration of about 1-20, preferably of about2-15, particularly preferably about 3-10 and especially of about 4-8,mg/kg of the subject's body weight. Also conceivable are combinations ofthe compounds of the invention in freely combinable concentrationproportions of the ranges mentioned.

[0019] The invention also includes compositions comprising at least onecompound of the aforementioned type for regulating the biologicalfunction of proteins which themselves are controlled in terms of theiractivity by protein kinases which in turn are inhibited by the compoundsof the invention (signal transduction).

[0020] It is additionally possible for the compositions of the inventionto comprise additional substances which are necessary or suitable forbetter processing or administration to the patient. These substances,just like the processes for producing the compositions of the invention,are routine laboratory practice and are therefore not explained indetail here.

[0021] The present invention relates to the use of the compounds of theaforementioned type for producing compositions for inhibiting theuncontrolled proliferation and/or inducing the apoptosis of cells and/orfor inhibiting angiogenesis and/or lymphangiogenesis. Likewise includedis the use of the compounds of the invention for the treatment ofangiogenesis- and/or lymphangiogenesis-dependent disorders (Table 2)and/or filariasis.

[0022] The following examples serve to illustrate the present inventionbut do not have a limiting effect on the invention:

[0023] 1) Structure and Synthesis of MAE87, MAE106 and MAZ51

[0024] General Method:

[0025] 10 mmol of oxoindole, 10 mmol of aldehyde and a few drops ofpiperidine are dissolved in 40 ml of ethanol. The reaction mixture isstirred at 90° C. for 5 hours. The desired product (E/Z mixture)precipitates during the reaction or after cooling to room temperature.The precipitate is filtered off, washed with ethanol and dried in vacuo.

[0026] The NMR spectra were recorded on a Bruker DRX500 spectrometer.The chemical shifts are stated in ppm (parts per million). The residualproton signal of the solvents serves as internal standard.High-resolution mass spectra were recorded using a Finnigan MAT MS 70mass spectrometer. The melting points are uncorrected.

[0027] 1a) 3-(2,4-Dihydroxybenzylidene)-1,3-dihydroindol-2-one (MAE87)

[0028] A mixture of 1.33 g of indole-2-one (10 mmol), 1.38 g of2,4-dihydroxybenzaldehyde (10 mmol) and 3 drops of piperidine arerefluxed in 40 ml of ethanol for 5 hours. The desired productprecipitates as a yellow solid during the reaction. After cooling toroom temperature, the product is filtered off, washed with ethanol anddried in vacuo. Yield 1.28 g (51%).

[0029] Melting point: 250° C. decomposition

[0030]¹H-NMR (500 MHz, DMSO-d₆):

[0031] δ [ppm]: 6.38 (dd, J=2 Hz, J=8.5 Hz, 1H); 6.44 (d, J=2 Hz, 1H);6.85 (m, 2H); 7.17 (t, J=7.5 Hz, 1H); 7.55 (d, J=8.3 Hz, 1H); 7.63 (d,J=7.6 Hz, 1H); 7.7 (s, 1H); 10.2 (br, 2H); 10.45 (s, 1H)

[0032]¹³C-NMR (125 MHz, DMSO-d₆):

[0033] δ [ppm]: 102.9; 107.4; 110.2; 113.1; 121.3; 122.2; 122.3; 123.5;129.2; 131.2; 133.4; 142.5; 159.2; 161.7; 169.7

[0034] HR-MS: C₁₅H₁₁NO₃: calc.: m/z=253.0739 found: m/z 253.0744

[0035] 1b) 3-(3-Fluoro-4-methoxybenzylidene)-1,3-dihydroindole-2-one(MAE106)

[0036] A mixture of 1.33 g of indole-2-one (10 mmol), 1.54 g of3-fluoro-4-methoxybenzaldehyde (10 mmol) and 3 drops of piperidine arerefluxed in 40 ml of ethanol for 5 hours. The desired productprecipitates as a yellow crystalline solid during the reaction. Aftercooling to room temperature, the product is filtered off, washed withethanol and dried in vacuo. Yield 2.2 g (82%).

[0037] Melting point: 220° C.

[0038]¹H-NMR (500 MHz, DMSO-d₆ E/Z isomers:

[0039] δ [ppm]: 3.92 (s, 3H); 6.83 (d, J=7.2 Hz, 0.7H); 6.87 (m, 0.7H);6.98 (t, J=7.6 Hz, 0.7H); 7.18-7.32 (m, 2H); 7.54 (s, 0.3H); 7.6 (m,1H); 7.65 (d, J=7.6 Hz, 0.6H); 7.74 (s, 0.7H); 8.0 (d, J=8.3 Hz, 0.7H);8.80 (dd, J=2 Hz, J=13 Hz, 0.6H); 10.65 (br, 1H)

[0040]¹³C-NMR (125 MHz, DMSO-d₆):

[0041] δ [ppm]: 109.8; 110.6; 113.6; 114.3; 117.4; 117.6; 118.9; 119.0;119.9; 121.3; 121.5; 121.6; 122.6; 125.5; 125.7; 121.1; 127.2; 127.5;127.6; 127.7; 127.8; 129.1; 130.5; 121.1; 135.1; 136.2; 140.9; 143.3;148.7; 148.8; 149.5; 149.6; 150.0; 150.6; 151.9; 152.5; 167.8; 169.2

[0042] HR-MS: C₁₆H₁₂NO₂F: calc.: m/z=298.9946 found: m/z 298.9954

[0043] 1c)3-(4-Dimethylaminonaphthalen-1-ylmethylene)-1,3-dihydroindol-2-one(MAZ51)

[0044] A mixture of 1.33 g of indole-2-one (10 mmol), 1.99 g of4-dimethylamino-1-naphthaldehyde (10 mmol) and 3 drops of piperidine arerefluxed in 40 ml of ethanol for 5 hours. The desired productprecipitates as an orange solid during the reaction. After cooling toroom temperature, the product is filtered off, washed with ethanol anddried in vacuo. Yield 2.67 g (85%).

[0045]¹H-NMR (500 MHz, DMSO-d₆):

[0046] δ [ppm]: 2.90 (s, 6H; (CH₃)₂) 6.74 (dt, J=1 Hz, J=7.5 Hz, 1H);6.87 (d, J=7.5 Hz, 1H); 7.15 (m, 2H); 7.24 (d, J=8 Hz, 1H); 7.59 (m,2H); 7.83 (d, J=8 Hz, 1H); 7.93 (m, 1H); 8.06 (s, 1H); 8.23 (m, 1H);10.64 (br, 1H, —NH).

[0047]¹³C-NMR (125 MHz, DMSO-d₆):

[0048] δ [ppm]: 45.1; 110.4; 113.4; 121.4; 121.7; 122.7; 125.2; 125.3;125.4; 126; 127.4; 128.1; 128.2; 128.3; 130.2; 132.8; 134.1; 143.2;153.1; 169

[0049] HR-MS: C₂₁H₁₈N₂O: calc.: m/z=314.1419 found: m/z 314.1419

[0050] Melting point: 250° C. decomposition

[0051] 2) MAE87, MAE106 and MAZ51 Inhibit a Large Number of ReceptorTyrosine Kinases in the In Vitro Tyrosine Kinase Assay. Inhibitor KinaseName Conc. VEGFR-3 VEGFR-2 Tie2 EGFR ErbB2 IGF1R FGFR1 MAE87  1 μg/ml −9−46 +1 −70 −45 −63 −50 10 μg/ml −74 −77 −65 −86 −84 −94 −94 MAE106  1μg/ml +1 +21 −5 −5 +4 −10 +5 10 μg/ml −57 −58 −28 −64 −41 −71 −1 MAZ51 1 μg/ml −4 −8 −7 −25 −11 −10 +1 10 μg/ml −35 −74 −27 −58 −39 −68 −5

[0052] The inhibition of various receptor tyrosine kinases in vitro byMAE87, MAE106 and MAZ51 was investigated. The inhibitors wereinvestigated in 2 different concentrations (1 μg/ml and 10 μg/ml). Theinhibition is stated in %. “+”=stimulation of kinase activity relativeto the control,

[0053] “−”=inhibition of kinase activity relative to the control.

[0054] The activity of the RTK inhibitors were determined with the aidof an ELISA. In this assay, the corresponding kinases were employed asrecombinant GST fusion proteins (glutathione S-transferase). SyntheticpolyGlu, Tyr 4:1 is used as acceptor. The ability of differentconcentrations of the test substances to inhibit the RTK-mediatedphosphorylation of the abovementioned acceptor was assessed. All thetests were carried out in duplicate.

[0055] ELISA plates were coated with 0.2 mg/ml of polyGlu, Tyr 4:1 in100 mM bicarbonate buffer (pH 9.6) overnight. This solution was removedand the microtiter plates were washed twice with TBS buffer (10 mMTris-HCl, pH 8.1, 100 mM NaCl) and then blocked with 5% BSA/TBS for 30min. 50 μl of test substance (2 or 20 μg/ml in 10% DMSO), 25 μl of GSTkinase in 4× kinase buffer (200 mM HEPES, 100 mM NaCl, 80 μM Na₃VO₄ and0.04% BSA) were introduced. The reaction was started by adding 25 μl of160 μM ATP (in 40 mM MnCl₂) as substrate of the kinases. The finalconcentrations of the test compounds is thus 1 or 10 μg/ml in 5% DMSO.The GST fusion proteins were employed in the following concentrations.VEGFR-2 50 NG/well, VEGFR-3 300 ng/well, Tie2 300 ng/well, EGFR 50ng/well, ErbB2 200 ng/well, IGF-R 50 ng/well, FGFR1 200 ng/well. Thereaction mixture was incubated at 30° C. for 90 min. The kinase reactionwas stopped by adding 50 μl of 30 mM EDTA/well. The microtiter plateswere washed twice with 0.05% Tween20/TBS. Anti-phosphotyrosine antibody(1:500) was added in 0.05% Tween20/TBS (with 0.5% BSA, 0.025% skimmedmilk powder and 100 μM Na₃VO₄) and incubated at 37° C. for 1 h. Themicrotiter plates were washed three times with 0.05% Tween20/TBS. TheHRP-conjugated detection autibody (1:1000) was added in 0.05%Tween20/TBS (with 0.5% BSA, 0.025% skimmed milk powder and 100 μMNa₃VO₄) and incubated at 37° C. for 1 h. The microtiter plates werewashed three times with 0.05% Tween20/TBS. ABTS(2,2′-azino-di-(3-ethylbenz-thiazoline-6-sulfonate)) substrate (RocheDiagnostics GmbH, Mannheim) was added. The OD was determined at 405 nmusing an ELISA reader.

[0056] 3) MAE87, MAE106 and MAZ51 Inhibit the Ligand-inducedAutophosphorylation of the RTKs VEGFR-2 and VEGFR-3

[0057] VEGFR-2- and VEGFR-3-expressing PAE cells (pig aorta endothelcells) were incubated with 0.5 μM, 5 μM or 50 μM of the appropriate testcompound. The cells were then stimulated with VEGF (VEGFR-2) or VEGF-C(VEGFR-3) or without growth factor as negative control. The cells wereharvested after 15 min and immunoprecipitated with VEGFR-2 or VEGFR-3.The immunoprecipitates were blotted after electrophoresis andinvestigated with anti-phosphotyrosine antibody. The blots were detachedand investigated with VEGFR-2 or VEGFR-3 antibody to check the loading.At 5 μM, MAZ51 selectively inhibits VEGFR-3.

[0058] PAE/VEGFR-2 or PAE/VEGFR-3 were seeded in 15 cm tissue culturetissues and cultivated to 50% confluence. The cells were then starved inserum-free medium (with 0.2% BSA) for 16-24 h (PAE/VEGFR-3) or 72 h(PAE/VEGFR-3). After preincubation with 5 ml of 1 mM serum-free medium(1 mM Na₃VO₄) and various inhibitor concentrations for 30-60 min, thecells were stimulated at 37° C. for 5 min (VEGFR-3) or 8 min (VEGFR-2).The cells were then rapidly washed twice with ice-cold PBS (with 1 mMNa₃VO₄) and lysed with ice-cold modified RIPA buffer (30 mM Tris-HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) Triton X100, 0.5% (w/v) sodiumdeoxycholate, 10 mM NaF) freshly prepared with 1 mM PMSF, 0.1 U/mlaprotinin, 10 ng/ml leupeptin and 5 mM Na₃VO₄. The cells were harvestedfrom the plate with the aid of a rubber policeman and collected incentrifuge tubes on ice. The lysates were solubilized by forcing througha syringe with a 25 G needle, and were centrifuged at 4° C./13 000 rpmfor 15 min to remove insoluble constituents.

[0059] The clear lysates were incubated with 4 μg of anti-VEGFR-2(C-1158, Santa Cruz) or anti-VEGFR-2 antibody (M20, Santa Cruz) at 4° C.overnight.

[0060] The receptor-antibody complex was precipitated by adding 30 μl ofprotein A-Sepharose (Amersham)/tube and incubating at 4° C. for afurther 2 hours.

[0061] The Sepharose were then centrifuged at 4° C. for 1 min and washedthree times with cold washing buffer buffer (30 mM Tris-HCl, pH 7.4, 150mM NaCl, 1 mM EDTA, 0.5% (v/v) Triton X100, 0.5% (w/v) sodiumdeoxycholate, 10 mM NaF) freshly prepared with 1 mM PMSF, 0.1 U/mlaprotinin, 10 ng/ml leupeptin and 5 mM Na₃VO₄. The remainder of thewashing buffer was removed by aspiration through a syringe provided witha 27 G needle. The Sepharose beads were resuspended in 50 μl of SDSloading buffer, boiled and loaded onto a 6% agarose gel. After theelectrophoretic fractionation, the proteins were fixed on an appropriatemembrane by Western blotting. The blots were investigated first with theaid of an anti-phosphotyrosine antibody (RC20:HRPO, Becton Dickinson)and then the first antibody was removed again in order to investigatethe blot for the protein loading with specific anti-receptor antibodies.The first antibody was stripped off by shaking the membranes withstripping buffer (62.5 mM Tris, pH —6.8, 2% SDS, 0.75%2-mercaptoethanol) at 55° C. for 20 min. The membranes were then washedtwice with TBST for 2 min each time and blocked and incubated as usual.

[0062] The result is depicted in FIG. 1.

[0063] 4) MAE87, MAE106 and MAZ51 Inhibit Endothelial Cell Proliferation

[0064] Human umbilical vein endothelial cells (HUVEC) underwentserum-starved cultivation for 24 h. The cells were then preincubatedwith the appropriate compounds for 2 h and subsequently cultivated inthe presence of the inhibitors with VEGF (MAE87) or bFGF (MAE106, MAZ51)for a further 24 h. The cells were then incubated with ³H-thymidine. Theamount of radioactivity incorporated into the cellular DNA was measured.All the experiments were carried out in triplicate.

[0065] HUVE cells were seeded in 100 μl/well (10⁵ cells/ml) in 96-wellculture plates and left to rest overnight. The cells were then starvedwith 50 μl of serum-free medium/well for 24 h. Subsequently, 50 μl ofvarious concentrations of the test substances in serum-free medium(contains 2% DMSO) were added and incubated at 37° C. for 2 h. Themedium containing the test compounds as removed and replaced by 50 μl offresh medium with FGF (12.5 ng/ml) or VEGF (100 ng/ml). Fresh dilutionsf the doubly concentrated test substances in serum-free medium with 2%DMSO were added to the cells in 50 μl/well. The final concentration ofDMSO was always 1%. After 24 h, ³H-thymidine (1 μCi/well) was added, andthe cells were incubated for a further 4-6 h. To analyze theincorporated radioactivity, the cells were trypsinized for 30 min andharvested with the aid of a harvester 96 cell (Tomtec) and fixed on aglass fiber membrane filter. The immobilized radioactivity wasquantified with the aid of a MicroBeta TriLux liquid scintillationluminescence counter. The result is depicted in FIG. 2.

[0066] 5) MAE87, MAE106 and MAZ51 Inhibit Angiogenesis in the CAM Assay

[0067] The chorioallantoic membrane (CAM) assay was carried out asdescribed (Wernert et al.) with chick embryos 5 days afterfertilization. Methylcellulose disks (diameter 2 mm) with 100 μg ofinhibitor were applied to the CAMs. Evaluation took place on day 7.Representative results for MAE106 are shown in FIG. 3. A. negativecontrol B. MAE 106-treated egg. The highly branched network of bloodvessels shown by the negative control is highly underdeveloped in theMAE 106-treated egg.

[0068] 6) MAE87, MAE106 and MAZ51 Inhibit the Proliferation of TumorCells

[0069] Cells of the rat tumor cell line (Nestl et al., 2001, CancerResearch 61: 1569-1577) were cultivated in the presence of 1% DMSO(solvent control), 2.5 μM or 10 μM of the indolinone for 24 h. Tritiatedthymidine was added to the medium during the last 4-6 h of incubation.The cells were harvested and the amount of radioactivity incorporatedinto the DNA was quantified.

[0070] The data are stated in percent relative to the proliferation ofthe cells treated only with DMSO (% relative to control; see FIG. 4)).MAZ51 had the greatest inhibitory effect.

[0071] 7) MAE87, MAE106 and MAZ51 Induced Apoptosis in Endothelial Cellsand Tumor Cells

[0072] Human endothelial cells (HDMEC) and rat pancreatic tumor cells(1AS) were resuspended (10⁵ cells/ml). 50 μl of cell suspension wereseeded in 96-well cell culture plates and incubated at 37° C. for 24 h.The cells were then incubated with the stated concentrations of thevarious inhibitors for a further 24 h. The pro-apoptotic effect of thecompounds was determined using the Cell Death Detection ELISA^(plus) kit(Roche Diagnostics GmbH, Mannheim) in accordance with the manufacturer'sstatements. The kit contains a photometric enzyme immunoassay forqualitative and quantitative in vitro determination of cytoplasmichistone-associated DNA fragments (mono- and oligo-nucleosomes) releasedthrough active cell death (apoptosis). The proportion of inducedapoptosis can be quantified by determining the optical density at 405nm. The measurements relative to the untreated cells are plotted againstthe inhibitor concentration. The results in FIG. 5 show that MAZ51 is apotent activator of apoptosis both in endothelial cells and tumor cells.

[0073] Cells of the rat tumor cell line (Nestl et al., 2001, CancerResearch 61: 1569-1577) were cultivated in the presence of 1% DMSO(solvent control), 2.5 μM or 10 μM of the indolinone for 24 h. Apoptosiswas quantified with the aid of anti-DNA peroxidase antibody. The amountof dye produced in the chromogenic reaction catalyzed by the peroxidasewas measured by photometry at 405 nm and correlated with the amount ofapoptosis-associated cytoplasmic mono- and oligonucleosomes. The dataare stated in % apoptosis relative to the cells of the solvent control(% relative to control) (FIG. 6). MAZ51 is the most potent apoptosisinducer.

[0074] 8) MAZ51 Inhibits the Growth of 1AS and MT450 Rat Carcinomas InVivo.

[0075] 1AS cells (5×10⁵) were injected subcutaneously into 2 groups ofBDX rats (8 rats per group). One group was subsequently injected with100 μl of DMSO/animal per day up to the end of the experiment. The othergroup was injected with 100 μl of MAZ51 (10 mg/ml in DMSO)/animal,equivalent to 4-5 mg/kg, each day. The tumor volume was measurednormally measured regularly after the injection of the tumor cells. Ascan be seen from FIG. 7, the growth of AS1 tumors in vivo isconsiderably inhibited through treatment with MAZ51.

[0076] MT450 rat mammary carcinoma cells were injected subcutaneouslyinto Wistar Furth rats. Treatment with the active substance MAZ51 at 8mg/kg/day in 100% DMSO or solvent control (100% DMSO) was started theday after injection of the tumor cells. MAZ51 or only solvent wasinjected intraperitoneally each day. Each test group comprised 8animals. The tumors were measured every 4-5 days. The average tumorvolumes are indicated in FIG. 8.

KEY TO THE TABLES AND FIGURES

[0077] Table 1: Survey of activation mutations of various receptortyrosine kinases and the diseases induced thereby.

[0078] Table 2: Survey of antiogenesis-dependent diseases

[0079]FIG. 1: Western blot of immunoprecipitations (i.p.) of VEGFR-2 andVEGFR-3 from PAE cells treated with MAE87, MAE106 and MAZ51. (−)represents the control (unstimulated cells); (+) are growthfactor-stimulated cells treated without or with 0.5 μM, 5 μM or 50 μM ofthe appropriately indicated inhibitor (MAE87, MAE106 or MAZ51). Theantibody probes (probe) employed for the immunoprecipitation is eitheranti-phosphotyrosine antibody (anti-phospho-Y), anti-VEGFR-2-antibody(anti-VEGFR-2) or anti-VEGFR-3-antibody (anti-VEGFR-3).

[0080]FIG. 2: Measurement of the (³H)-thymidine incorporation into humanendothelial cells (HUVEC) as a function of the concentration ofinhibitors employed (MAE87, MAE106 and MAZ51). The inhibition of cellproliferation by MAE87, MAE106 and MAZ51 is dose-dependent in this case.

[0081]FIG. 3: Inhibition of angiogenesis in a chorioallantoic membrane(CAM). A: sham-treated cells (control). B: Mae106-treated cells of theCAM.

[0082]FIG. 4: Measurement of the (3H)-thymidine incorporation (in %)into rat tumor cell lines (ASML, 1AS, G, AT6.1, MTLN3, MTLY, NM081 andMT450) as a function of the concentration of inhibitors employed. Theinhibition of cell proliferation by MAE87, MAE106 and MAZ51 isdose-dependent.

[0083]FIG. 5: Nucleosome ELISA test (Cell Death Detection ELISA). Theextent of dead cells of the respective cell line is depicted (asrelative absorption at 405 nm) in relation to the use of the inhibitorsMAE87, MAE106 and MAZ51 and MAZ51-2. MAZ51 is the most effective atinducing cell death in human endothelial cells (HDMEC) and the ratpancreatic cell line 1AS.

[0084]FIG. 6: Nucleosome ELISA test (Cell Death Detection ELISA). Theextent of dead cells of the respective rat tumor cell line is depicted(apoptosis in %) in relation to the use of the inhibitors MAE87, MAE106and MAZ51. MAE87, MAE106 and MAZ51 induce cell death in a number of ratcarcinoma cell lines.

[0085]FIG. 7: Depiction of the tumor volume as a function of the numberof days after tumor cell injection depending on the treatment of the 1AStumor cells with MAZ51. The investigated rats show that tumor growth isinhibited in vivo after treatment of 1AS tumor cells with MAZ51.

[0086]FIG. 8: Depiction of the tumor volume as a function of the numberof days after tumor cell injection depending on treatment of the MT450tumor cells with MAZ51 and a DMSO solution (negative control). Theinvestigated rats show that tumor growth of MT450 tumor cells isinhibited in vivo after treatment with MAZ51. +/−SE means the standarddeviation of the tumor volume from the average of 8 investigated ratsper batch.

1. A compound which inhibits the activity of protein kinases proteinkinases which are involved in uncontrolled cell proliferation and/ordisordered apoptosis of cells and/or angiogenesis- and/orlymphangiogenesis-dependent disorders and/or filariasis and represents aderivative, substituted on the C3 atom, of indolin-2-one, selected fromthe group of compounds of the general structural formula

or salts of the compounds I)-III).
 2. The compound as claimed in claim1, wherein it inhibits the activity of receptor tyrosine kinases.
 3. Thecompound as claimed in claim 1, wherein it inhibits the activity ofreceptor tyrosine kinases from the group of VEGFR-3 (vascularendothelial cell growth factor receptor), VEGFR-2, Tie2, EGFR (epidermalgrowth factor receptor), ErbB2, IGFR and FGFR1 (fibroblast growth factorreceptor).
 4. The compound as claimed in claim 1, wherein it inhibitsthe activity of the cell surface receptor VEGFR-3.
 5. A composition forinhibiting uncontrolled proliferation and/or inducing apoptosis of cellscomprising at least one compound as claimed in claim
 1. 6. Thecomposition as claimed in claim 5 for inhibiting angiogenesis and/orlymphangiogenesis.
 7. The composition as claimed in claim 5 for thetreatment of angiogenesis-dependent and/or lyrnphangiogenesis-dependentdiseases and/or filariasis.
 8. The composition as claimed in claim 5,wherein it comprises at least one compound as claimed in claim 1 in aconcentration of about 1-20, preferably of about 2-15, particularlypreferably of about 3-10 and in particular of about 4-8, mg/kg of thesubject's body weight.
 9. The use of the compounds as claimed in claim 1for producing compositions for inhibiting uncontrolled proliferationand/or induction apoptosis of cells and/or for inhibiting angiogenesisand/or lymphangiogenesis.
 10. The use of compounds as claimed in claim 1for the treatment of angiogenesis-dependent and/orlymphangiogenesis-dependent diseases and/or filariasis.